T cell receptors

ABSTRACT

The present invention relates to T cell receptors (TCRs) which bind the HLA-A2 restricted FMNKFIYEI (158-166) peptide epitope derived from α Fetoprotein (AFP). Certain preferred TCRs of the invention demonstrate excellent binding characteristics and specificity profiles for this AFP epitope. T cell receptors of the invention may comprise at least one TCR alpha chain variable domain and/or at least one TCR beta chain variable domain, the alpha chain variable domain which may comprise an amino acid sequence that has at least 90% identity to the sequence of amino acid residues 1-112 of SEQ ID No: 2, and/or the beta chain variable domain which may comprise an amino acid sequence that has at least 90% identity to the sequence of amino acid residues 1-112 of SEQ ID No: 3.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a divisional of U.S. application Ser. No. 15/006,224filed Jan. 26, 2016, now allowed, which is a continuation-in-partapplication of international patent application Serial No.PCT/GB2014/052199 filed Jul. 18, 2014, which published as PCTPublication No. WO 2015/011450 on Jan. 29, 2015, which claims benefit ofUnited Kingdom patent application Serial No. 1313377.2 filed Jul. 26,2013.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appin cited documents”) and all documents cited orreferenced in the appin cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 9, 2015, isnamed 43784_00_2005 SL.txt and is 47,533 bytes in size.

FIELD OF THE INVENTION

The present invention relates to T cell receptors (TCRs) which bind theHLA-A2 restricted FMNKFIYEI (158-166) peptide epitope derived from αFetoprotein (AFP). Certain preferred TCRs of the invention demonstrateexcellent binding characteristics and specificity profiles for this AFPepitope.

BACKGROUND OF THE INVENTION

AFP is expressed during foetal development and is the main component offoetal serum. During development the protein is produced at very highlevels by the yolk sac and liver and is later repressed. AFP expressionis frequently reactivated in hepatocellular carcinoma (Butterfield etal. J Immunol., 2001, Apr. 15; 166(8):5300-8) and high levels of theprotein are used as a diagnostic marker for the disease.

Hepatocellular carcinoma has one of the lowest reported 5 year survivalrate of all malignancies in the US, global annual incidence is 1.2million and is likely to increase due to the pandemic of Hepatitis B andC. Treatment typically involves surgery, however this is only beneficialif carried out in the early stages of the disease. New treatments aretherefore desirable.

There are four known epitopes derived from AFP: AFP158, AFP137, AFP325and AFP542 (Butterfield et al. J Immunol., 2001, Apr. 15; 166(8):5300-8and Butterfield et al. Cancer Res. 1999, 59: 3134-3142). The HLA-A2restricted AFP158 peptide FMNKFIYEI (SEQ ID No: 1) provides a suitabletarget for novel immunotherapeutic interventions; this peptide isnaturally processed and has been eluted from HepG2 (HLA-A2 positive)liver carcinoma lines and detected by mass spectrometry (Butterfield etal. J Immunol., 2001, Apr. 15; 166(8):5300-8). T cell clones have beenraised against AFP158 (and AFP137) (Pichard et al. J Immunother. 2008April; 31(3):246-53). However, T cell receptors which recognize thispeptide have not been reported.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The present invention relates to a non-naturally occurring and/orpurified and/or engineered T cell receptor (TCR) having the property ofbinding to FMNKFIYEI (SEQ ID No: 1) HLA-A2 complex and which maycomprise at least one TCR alpha chain variable domain and/or at leastone TCR beta chain variable domain, the alpha chain variable domainwhich may comprise an amino acid sequence that has at least 90% identityto the sequence of amino acid residues 1-112 ofTRAV12-2*02/TRAJ41*01/TRAC, the extracellular sequence of the parentalAFP TCR alpha chain is given in FIG. 1 (SEQ ID No: 2), and/or the betachain variable domain which may comprise an amino acid sequence that hasat least 90% identity to the sequence of amino acid residues 1-112 ofTRBV9*01/TRBD2/TRBJ2-7*01/TRBC2, the extracellular sequence of theparental AFP TCR alpha chain is given in FIG. 2 (SEQ ID No: 3).

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product. It may be advantageous in thepractice of the invention to be in compliance with Art. 53(c) EPC andRule 28(b) and (c) EPC. All rights to explicitly disclaim anyembodiments that are the subject of any granted patent(s) of applicantin the lineage of this application or in any other lineage or in anyprior filed application of any third party is explicitly reservedNothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1 (SEQ ID No: 2) gives the amino acid sequence of the extracellularpart of the alpha chain of the parental AFP-specific TCR with gene usageTRAV12-2*02/TRAJ41*01/TRAC.

FIG. 2 (SEQ ID No: 3) gives the amino acid sequence of the extracellularpart of the beta chain of the parental AFP-specific TCR with gene usageTRBV9*01/TRBD2/TRBJ2-7*01/TRBC2.

FIG. 3 (SEQ ID No: 4) gives the amino acid sequence of the alpha chainof a soluble TCR (referred to herein as the “reference TCR”). Thesequence is the same as that of FIG. 1 (SEQ ID No: 2) except that acysteine (bold and underlined) is substituted for T159 of SEQ ID No: 2(i.e. T48 of the TRAC constant region).

FIG. 4 (SEQ ID No: 5) gives the amino acid sequence of the beta chain ofa soluble TCR (referred to herein as the “reference TCR”). The sequenceis the same as that of FIG. 2 (SEQ ID No: 3) except that a cysteine(bold and underlined) is substituted for S169 of SEQ ID No: 3 (i.e. S57of the TRBC2 constant region) and A187 is substituted for C187 and D201is substituted for N201.

FIG. 5 (SEQ ID Nos: 6-20) gives the amino acid sequence of the mutatedalpha chains which may be present in TCRs of the invention. The CDRregions are underlined and amino acid changes relative to the parentalAFP TCR are shaded.

FIG. 6 (SEQ ID No: 21) and (SEQ ID No: 22) gives the DNA sequencesencoding the TCR alpha and beta chains shown in FIGS. 3 and 4respectively

FIG. 7 (SEQ ID No: 23) gives the DNA sequence for the parental AFP TCRgene (alpha chain-2A-beta chain construct with the Porcine teschovirus-12A sequence bold and underlined) for transduction of T-cells.

FIG. 8 (SEQ ID No: 24) gives the amino acid sequence of the parental AFPTCR for T-cell transduction produced from the DNA sequence of FIG. 7.The Porcine teschovirus-1 2A sequence is bold and underlined.

FIG. 9 shows the results of an ELISPOT assay in which IFN-γ release ofAFP TCR-transduced T-cells in response to a range of target cells wasassessed.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention, there is provided anon-naturally occurring and/or purified and/or engineered T cellreceptor (TCR) having the property of binding to FMNKFIYEI (SEQ IDNo: 1) HLA-A2 complex and which may comprise at least one TCR alphachain variable domain and/or at least one TCR beta chain variabledomain,

-   -   the alpha chain variable domain which may comprise an amino acid        sequence that has at least 90% identity to the sequence of amino        acid residues 1-112 of SEQ ID No: 2, and/or    -   the beta chain variable domain which may comprise an amino acid        sequence that has at least 90% identity to the sequence of amino        acid residues 1-112 of SEQ ID No: 3.

In some embodiments of the invention, the alpha chain variable domainhas at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to amino acid residues 1 to 112 of SEQ ID No: 2.

The beta chain variable domain may have at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acidresidues 1 to 112 of SEQ ID No: 3.

TCRs are described using the International Immunogenetics (IMGT) TCRnomenclature, and links to the IMGT public database of TCR sequences.Native alpha-beta heterodimeric TCRs have an alpha chain and a betachain. Broadly, each chain may comprise variable, joining and constantregions, and the beta chain also usually contains a short diversityregion between the variable and joining regions, but this diversityregion is often considered as part of the joining region. Each variableregion may comprise three CDRs (Complementarity Determining Regions)embedded in a framework sequence, one being the hypervariable regionnamed CDR3. There are several types of alpha chain variable (Vα) regionsand several types of beta chain variable (Vβ) regions distinguished bytheir framework, CDR1 and CDR2 sequences, and by a partly defined CDR3sequence. The Vα types are referred to in IMGT nomenclature by a uniqueTRAV number. Thus “TRAV21” defines a TCR Vα region having uniqueframework and CDR1 and CDR2 sequences, and a CDR3 sequence which ispartly defined by an amino acid sequence which is preserved from TCR toTCR but which also includes an amino acid sequence which varies from TCRto TCR. In the same way, “TRBV5-1” defines a TCR Vβ region having uniqueframework and CDR1 and CDR2 sequences, but with only a partly definedCDR3 sequence.

The joining regions of the TCR are similarly defined by the unique IMGTTRAJ and TRBJ nomenclature, and the constant regions by the IMGT TRACand TRBC nomenclature.

The beta chain diversity region is referred to in IMGT nomenclature bythe abbreviation TRBD, and, as mentioned, the concatenated TRBD/TRBJregions are often considered together as the joining region.

The α and β chains of αβ TCR's are generally regarded as each having two“domains” or “regions”, namely variable and constant domains/regions.The terms “domain(s)” and “region(s)” are used interchangeably herein.The variable domain consists of a concatenation of variable region andjoining region. In the present specification and claims, the term “TCRalpha variable domain” therefore refers to the concatenation of TRAV andTRAJ regions, and the term TCR alpha constant domain refers to theextracellular TRAC region, or to a C-terminal truncated TRAC sequence.Likewise the term “TCR beta variable domain” refers to the concatenationof TRBV and TRBD/TRBJ regions, and the term TCR beta constant domainrefers to the extracellular TRBC region, or to a C-terminal truncatedTRBC sequence.

The unique sequences defined by the IMGT nomenclature are widely knownand accessible to those working in the TCR field. For example, they canbe found in the IMGT public database. The “T cell Receptor Factsbook”,(2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8 alsodiscloses sequences defined by the IMGT nomenclature, but because of itspublication date and consequent time-lag, the information thereinsometimes needs to be confirmed by reference to the IMGT database.

SEQ ID Nos: 2 and 3 are, respectively, the alpha and beta chainextracellular sequences of what is referred to herein as the “parental”AFP TCR. The parental AFP TCR has the following alpha and beta chainusage:

Alpha chain: TRAV12-2*02/TRAJ41*01/TRAC (the extracellular sequence ofthe parental AFP TCR alpha chain is given in FIG. 1 (SEQ ID No: 2). TheCDRs are defined by amino acids 27-32 (CDR1), 50-55 (CDR2) and 90-101(CDR3) of SEQ ID NO: 2.

Beta chain: TRBV9*01/TRBD2/TRBJ2-7*01/TRBC2 (the extracellular sequenceof the parental AFP TCR alpha chain is given in FIG. 2 (SEQ ID No: 3).The CDRs are defined by amino acids 27-31 (CDR1), 49-54 (CDR2) and92-102 (CDR3) of SEQ ID NO: 3.

(Note, the terms ‘*01’ and ‘*02’ indicate there is more than one allelicvariant for this sequence, as designated by IMGT nomenclature, and thatit is the *01/*02 variant which is present in the TCR clone referred toabove. Note also that the absence of a “*” qualifier means that only oneallele is known for the relevant sequence.)

For the purpose of providing a reference TCR against which the bindingprofile of mutated TCRs of the invention may be compared, it isconvenient to use the soluble TCR having the extracellular sequence ofthe AFP TCR alpha chain given in FIG. 3 (SEQ ID No: 4) and theextracellular sequence of the AFP TCR beta chain given in FIG. 4 (SEQ IDNo: 5). That TCR is referred to herein as the “the reference TCR” or“the reference AFP TCR”. Note that SEQ ID No: 4 is identical to theparental alpha chain extracellular sequence SEQ ID No: 2 except thatC159 has been substituted for T159 (i.e. T48 of TRAC). Likewise SEQ IDNo: 5 is identical to the parental beta chain extracellular sequence SEQID No: 3 except that C169 has been substituted for S169 (i.e. S57 ofTRBC2), A187 has been substituted for C187 and D201 has been substitutedfor N201. These cysteine substitutions relative to the parental AFPalpha and beta chain extracellular sequences enable the formation of aninterchain disulfide bond which stabilises the refolded soluble TCR, iethe TCR formed by refolding extracellular alpha and beta chains. Use ofthe stable disulfide linked soluble TCR as the reference TCR enablesmore convenient assessment of binding affinity and binding half-life.

TCRs of the invention may be transformed into T cells, rendering themcapable of destroying cells presenting AFP HLA-A2 complex, foradministration to a patient in the treatment process known as adoptivetherapy. For this purpose, it would be desirable if the TCRs had ahigher affinity and/or a slower off-rate for the peptide-HLA complexthan native TCRs specific for that complex. Dramatic increases inaffinity have been associated with a loss of antigen specificity in TCRgene-modified CD8 T cells, which could result in the nonspecificactivation of these TCR-transfected CD8 cells. Therefore, TCRs having asomewhat higher affinity and/or a slower off-rate for the peptide-HLAcomplex than native TCRs specific for that complex, but not adramatically higher affinity and/or dramatically slower off-rate for thepeptide-HLA complex than native TCRs, would be preferred for adoptivetherapy (see Zhao et al., (2007) J Immunol. 179: 5845-54; Robbins etal., (2008) J Immunol. 180: 6116-31; and WO2008/038002).

Embodiments of the invention include TCRs which are mutated relative tothe parental AFP TCR. Mutated TCRs may comprise an alpha chain variabledomain that includes a mutation in one or more of the amino acidscorresponding to: 31Q, 32S, 94D, 95S, 96G, 97Y, and 98A, with referenceto the numbering shown in SEQ ID No: 2. For example, the alpha chainvariable domain may have one or more of the following mutations:

Residue no. 31Q F Y 32S A 94D Q 95S N 96G S 97Y V 98A S

The numbering used above is the same as that shown in FIG. 1 (SEQ ID No:2)

The alpha chain variable domain may comprise an amino acid sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identity to residues 1-112 of any one of SEQ ID No: 6, SEQ ID No:7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No:12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ IDNo: 17, SEQ ID No: 18, SEQ ID No: 19 and SEQ ID No: 20, and preferably,the amino acid sequence also has at least 90% identity to residues 1-112of SEQ ID No: 2. The amino acids underlined in FIG. 5 may be invariant.

In one embodiment, the TCR may comprise an alpha chain variable domainwhich may comprise Q1 to H112 of SEQ ID No: 11, SEQ ID No: 12 or SEQ IDNo: 13, and/or a beta chain variable domain which may comprise D1 toT112 of SEQ ID NO: 3.

Mutations can be carried out using any appropriate method including, butnot limited to, those based on polymerase chain reaction (PCR),restriction enzyme-based cloning, or ligation independent cloning (LIC)procedures. These methods are detailed in many of the of the standardmolecular biology texts. For further details regarding polymerase chainreaction (PCR) and restriction enzyme-based cloning, see Sambrook &Russell, (2001) Molecular Cloning—A Laboratory Manual (3^(rd) Ed.) CSHLPress. Further information on ligation independent cloning (LIC)procedures can be found in Rashtchian, (1995) Curr Opin Biotechnol 6(1):30-6.

Also within the scope of the invention are phenotypically silentvariants of any TCR disclosed herein. As used herein the term“phenotypically silent variants” is understood to refer to those TCRswhich have a K_(D) and/or binding half-life for the FMNKFIYEI (SEQ IDNo: 1) HLA-A2 complex within the ranges of KDs and binding half-livesdescribed below. For example, as is known to those skilled in the art,it may be possible to produce TCRs that incorporate changes in theconstant and/or variable domains thereof compared to those detailedabove without altering the affinity for the interaction with theFMNKFIYEI (SEQ ID No: 1) HLA-A2 complex. Such trivial variants areincluded in the scope of this invention. Those TCRs in which one or moreconservative substitutions have been made also form part of thisinvention.

As will be obvious to those skilled in the art, it may be possible totruncate the sequences provided at the C-terminus and/or N-terminusthereof, by 1, 2, 3, 4, 5 or more residues, without substantiallyaffecting the binding characteristics of the TCR. All such trivialvariants are encompassed by the present invention.

Native TCRs exist in heterodimeric αβ or γδ forms. However, recombinantTCRs consisting of αα or ββ homodimers have previously been shown tobind to peptide MHC molecules. Therefore, the TCR of the invention maybe a heterodimeric αβ TCR or may be an αα or ββ homodimeric TCR.

For use in adoptive therapy, an αβ heterodimeric TCR may, for example,be transfected as full length chains having both cytoplasmic andtransmembrane domains. In certain embodiments TCRs of the invention mayhave an introduced disulfide bond between residues of the respectiveconstant domains, as described, for example, in WO 2006/000830.

TCRs of the invention, particularly alpha-beta heterodimeric TCRs, maycomprise an alpha chain TRAC constant domain sequence and/or a betachain TRBC1 or TRBC2 constant domain sequence. The alpha and beta chainconstant domain sequences may be modified by truncation or substitutionto delete the native disulfide bond between Cys4 of exon 2 of TRAC andCys2 of exon 2 of TRBC1 or TRBC2. The alpha and/or beta chain constantdomain sequence(s) may also be modified by substitution of cysteineresidues for Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2, the saidcysteines forming a disulfide bond between the alpha and beta constantdomains of the TCR.

TCRs of the invention may be in single chain format, for example see WO2004/033685. Single chain formats include αβ TCR polypeptides of theVα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, Vα-Cα-L-Vβ-Cβ types, whereinVα and Vβ are TCR α and β variable regions respectively, Cα and Cβ areTCR α and β constant regions respectively, and L is a linker sequence.In certain embodiments single chain TCRs of the invention may have anintroduced disulfide bond between residues of the respective constantdomains, as described in WO 2004/033685.

The TCRs of the invention have the property of binding the FMNKFIYEI(SEQ ID No: 1) HLA-A2 complex. Certain TCRs of the invention have beenfound to be highly suitable for use in adoptive therapy. Such TCRs mayhave a K_(D) for the complex of less than the parental AFP TCR, forexample from about 1 μM to about 21 μM and/or have a binding half-life(T½) for the complex in the range of from less than 0.5 seconds to about2 seconds. Increasing the binding affinity of a native TCR often reducesthe selectivity of the TCR for its peptide-MHC ligand, and this isdemonstrated in Zhao Yangbing et al., (J. Immunol, 2007 179: 9, p5845-5854). However, certain TCRs of the invention remain selective forthe FMNKFIYEI HLA-A2 complex, despite, in some embodiments, havinghigher binding affinity than the parental AFP TCR (see Example 6).

Binding affinity (inversely proportional to the equilibrium constantK_(D)) and binding half-life (expressed as T½) can be determined by anyappropriate method. It will be appreciated that doubling the affinity ofa TCR results in halving the K_(D). T½ is calculated as ln 2 divided bythe off-rate (k_(off)). So doubling of T½ results in a halving ink_(off). K_(D) and k_(off) values for TCRs are usually measured forsoluble forms of the TCR, i.e. those forms which are truncated to removecytoplasmic and transmembrane domain residues. Therefore it is to beunderstood that a given TCR has an improved binding affinity for, and/ora binding half-life for the parental TCR if a soluble form of that TCRhas the said characteristics. Preferably the binding affinity or bindinghalf-life of a given TCR is measured several times, for example 3 ormore times, using the same assay protocol, and an average of the resultsis taken. In a preferred embodiment these measurements are made usingthe Surface Plasmon Resonance (BIAcore) method of Example 3 herein.

In a further aspect, the present invention provides nucleic acidencoding a TCR of the invention. In some embodiments, the nucleic acidis cDNA. In some embodiments, the invention provides nucleic acid whichmay comprise a sequence encoding an α chain variable domain of a TCR ofthe invention. In some embodiments, the invention provides nucleic acidwhich may comprise a sequence encoding a β chain variable domain of aTCR of the invention. In some embodiments, the invention providesnucleic acid which may comprise a sequence encoding both an α chainvariable domain of a TCR of the invention and a β chain variable domainof a TCR of the invention. The nucleic acid may be non-naturallyoccurring and/or purified and/or engineered.

In another aspect, the invention provides a vector which may comprisenucleic acid of the invention. Preferably the vector is a TCR expressionvector.

The invention also provides a cell harbouring a vector of the invention,preferably a TCR expression vector. The vector may comprise nucleic acidof the invention encoding in a single open reading frame, or twodistinct open reading frames, the alpha chain and the beta chainrespectively. Another aspect provides a cell harbouring a firstexpression vector which may comprise nucleic acid encoding the alphachain of a TCR of the invention, and a second expression vector whichmay comprise nucleic acid encoding the beta chain of a TCR of theinvention. Such cells are particularly useful in adoptive therapy. Thecells may be isolated and/or recombinant and/or non-naturally occurringand/or engineered.

Since the TCRs of the invention have utility in adoptive therapy, theinvention includes a non-naturally occurring and/or purified and/or orengineered cell, especially a T-cell, presenting a TCR of the invention.There are a number of methods suitable for the transfection of T cellswith nucleic acid (such as DNA, cDNA or RNA) encoding the TCRs of theinvention (see for example Robbins et al., (2008) J Immunol. 180:6116-6131). T cells expressing the TCRs of the invention will besuitable for use in adoptive therapy-based treatment of cancers such asthose of the pancreas and liver. As will be known to those skilled inthe art, there are a number of suitable methods by which adoptivetherapy can be carried out (see for example Rosenberg et al., (2008) NatRev Cancer 8(4): 299-308).

As is well-known in the art TCRs of the invention may be subject topost-translational modifications when expressed by transfected cells.Glycosylation is one such modification, which may comprise the covalentattachment of oligosaccharide moieties to defined amino acids in the TCRchain. For example, asparagine residues, or serine/threonine residuesare well-known locations for oligosaccharide attachment. Theglycosylation status of a particular protein depends on a number offactors, including protein sequence, protein conformation and theavailability of certain enzymes. Furthermore, glycosylation status (i.eoligosaccharide type, covalent linkage and total number of attachments)can influence protein function. Therefore, when producing recombinantproteins, controlling glycosylation is often desirable. Glycosylation oftransfected TCRs may be controlled by mutations of the transfected gene(Kuball J et al. (2009), J Exp Med 206(2):463-475). Such mutations arealso encompassed in this invention.

The TCR of the invention may be associated with a detectable label, atherapeutic agent or a PK modifying moiety.

Certain TCRs of the invention may be in soluble form (i.e. having notransmembrane or cytoplasmic domains). For stability, TCRs of theinvention, and preferably soluble heterodimeric TCRs, may have anintroduced disulfide bond between residues of the respective constantdomains, as described, for example, in WO 03/020763. Some soluble TCRsof the invention are useful for making fusion proteins which can be usedfor delivering detectable labels or therapeutic agents to antigenpresenting cells and tissues containing antigen presenting cells. Theymay therefore be associated (covalently or otherwise) with a detectablelabel (for diagnostic purposes wherein the TCR is used to detect thepresence of cells presenting the FMNKFIYEI (SEQ ID No 1) HLA-A2 complex;a therapeutic agent; or a PK modifying moiety (for example byPEGylation).

Detectable labels for diagnostic purposes include for instance,fluorescent labels, radiolabels, enzymes, nucleic acid probes andcontrast reagents.

Therapeutic agents which may be associated with the TCRs of theinvention include immunomodulators, radioactive compounds, enzymes(perforin for example) or chemotherapeutic agents (cis-platin forexample). To ensure that toxic effects are exercised in the desiredlocation the toxin could be inside a liposome linked to a TCR so thatthe compound is released slowly. This will prevent damaging effectsduring the transport in the body and ensure that the toxin has maximumeffect after binding of the TCR to the relevant antigen presentingcells.

Other suitable therapeutic agents include:

-   -   small molecule cytotoxic agents, i.e. compounds with the ability        to kill mammalian cells having a molecular weight of less than        700 Daltons. Such compounds could also contain toxic metals        capable of having a cytotoxic effect. Furthermore, it is to be        understood that these small molecule cytotoxic agents also        include pro-drugs, i.e. compounds that decay or are converted        under physiological conditions to release cytotoxic agents.        Examples of such agents include cis-platin, maytansine        derivatives, rachelmycin, calicheamicin, docetaxel, etoposide,        gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone,        sorfimer sodiumphotofrin II, temozolomide, topotecan,        trimetreate glucuronate, auristatin E vincristine and        doxorubicin;    -   peptide cytotoxins, i.e. proteins or fragments thereof with the        ability to kill mammalian cells. For example, ricin, diphtheria        toxin, pseudomonas bacterial exotoxin A, DNase and RNase;    -   radio-nuclides, i.e. unstable isotopes of elements which decay        with the concurrent emission of one or more of α or β particles,        or γ rays. For example, iodine 131, rhenium 186, indium 111,        yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213;        chelating agents may be used to facilitate the association of        these radio-nuclides to the high affinity TCRs, or multimers        thereof;    -   immuno-stimulants, i.e. immune effector molecules which        stimulate immune response. For example, cytokines such as IL-2        and IFN-γ, Superantigens and mutants thereof;    -   TCR-HLA fusions;    -   chemokines such as IL-8, platelet factor 4, melanoma growth        stimulatory protein, etc;    -   antibodies or fragments thereof, including anti-T cell or NK        cell determinant antibodies (e.g. anti-CD3, anti-CD28 or        anti-CD16);    -   alternative protein scaffolds with antibody like binding        characteristics complement activators;    -   xenogeneic protein domains, allogeneic protein domains,        viral/bacterial protein domains, viral/bacterial peptides.

For administration to patients, the TCRs or cells of the invention maybe provided in a pharmaceutical composition together with one or morepharmaceutically acceptable carriers or excipients. Cells in accordancewith the invention will usually be supplied as part of a sterile,pharmaceutical composition which will normally include apharmaceutically acceptable carrier. This pharmaceutical composition maybe in any suitable form, (depending upon the desired method ofadministering it to a patient). It may be provided in unit dosage form,will generally be provided in a sealed container and may be provided aspart of a kit. Such a kit would normally (although not necessarily)include instructions for use. It may include a plurality of said unitdosage forms.

The pharmaceutical composition may be adapted for administration by anyappropriate route, preferably a parenteral (including subcutaneous,intramuscular, or preferably intravenous) route. Such compositions maybe prepared by any method known in the art of pharmacy, for example bymixing the active ingredient with the carrier(s) or excipient(s) understerile conditions.

TCRs, pharmaceutical compositions, vectors, nucleic acids and cells ofthe invention may be provided in substantially pure form, for example atleast 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% pure.

Also provided by the invention are:

-   -   a non-naturally occurring and/or purified and/or engineered TCR        which binds the FMNKFIYEI peptide presented as a peptide-HLA-A2        complex, or a cell expressing and/or presenting such a TCR, for        use in medicine, preferably in a method of treating cancer. The        method may comprise adoptive therapy;    -   the use of a TCR which binds the FMNKFIYEI peptide presented as        a peptide-HLA-A2 complex, or a cell expressing and/or presenting        such a TCR, in the manufacture of a medicament for treating        cancer;    -   a method of treating cancer in a patient, which may comprise        administering to the patient a TCR which binds the FMNKFIYEI        peptide presented as a peptide-HLA-A2 complex, or a cell        expressing and/or presenting such a TCR.

It is preferred that the TCR which binds the FMNKFIYEI peptide presentedas a peptide-HLA-A2 complex is a TCR of the invention.

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis. The published documents mentionedherein are incorporated to the fullest extent permitted by law. Citationor identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1

Cloning of the Parental AFP TCR Alpha and Beta Chain Variable RegionSequences into pGMT7-Based Expression Plasmids

The reference AFP TCR variable alpha and TCR variable beta domains werePCR amplified from total cDNA isolated from an AFP T cell clone. In thecase of the alpha chain, an alpha chain variable region sequencespecific oligonucleotide A1gaattccatatgcaaaaagaagttgaacaaaattctggacccctc (SEQ ID No: 25) whichencodes the restriction site NdeI and an alpha chain constant regionsequence specific oligonucleotide A2ttgtcagtcgacttagagtctctcagctggtacacg (SEQ ID No: 26) which encodes therestriction site SalI are used to amplify the alpha chain variabledomain. In the case of the beta chain, a beta chain variable regionsequence specific oligonucleotide B1gaattccatatggattctggagttacacaaaccccaaagcacctg (SEQ ID No: 27) whichencodes the restriction site NdeI and a beta chain constant regionsequence specific oligonucleotide B2 tagaaaccggtggccaggcacaccagtgtggc(SEQ ID No: 28) which encodes the restriction site AgeI are used toamplify the beta chain variable domain.

The alpha and beta variable domains were cloned into pGMT7-basedexpression plasmids containing either Cα or Cβ by standard methodsdescribed in (Molecular Cloning a Laboratory Manual Third edition bySambrook and Russell). Plasmids were sequenced using an AppliedBiosystems 3730xl DNA Analyzer.

The DNA sequences encoding the TCR alpha chain cut with NdeI and SalIwere ligated into pGMT7+Cα vector, which was cut with NdeI and XhoI. TheDNA sequences encoding the TCR beta chain cut with NdeI and AgeI wasligated into separate pGMT7+Cβ vector, which was also cut with NdeI andAgeI.

Ligation

Ligated plasmids were transformed into competent E. coli strain XL1-bluecells and plated out on LB/agar plates containing 100 μg/ml ampicillin.Following incubation overnight at 37° C., single colonies are picked andgrown in 10 ml LB containing 100 μg/ml ampicillin overnight at 37° C.with shaking. Cloned plasmids were purified using a Miniprep kit(Qiagen) and the plasmids were sequenced using an Applied Biosystems3730xl DNA Analyzer.

FIGS. 3 and 4 show respectively the reference AFP TCR α and β chainextracellular amino acid sequences (SEQ ID Nos: 4 and 5 respectively)produced from the DNA sequences of FIG. 7 (SEQ ID No: 21) (SEQ ID No:22) respectively. Note that, relative to the parental TCR, cysteineswere substituted in the constant regions of the alpha and beta chains toprovide an artificial inter-chain disulphide bond on refolding to formthe heterodimeric TCR. The introduced cysteines are shown in bold andunderlined.

Example 2

Expression, Refolding and Purification of Soluble Parental AFP TCR

The expression plasmids containing the TCR α-chain and β-chainrespectively, as prepared in Example 1, were transformed separately intoE. coli strain BL21pLysS, and single ampicillin-resistant colonies weregrown at 37° C. in TYP (ampicillin 100 μg/ml) medium to OD₆₀₀ of˜0.6-0.8 before inducing protein expression with 0.5 mM IPTG. Cells wereharvested three hours post-induction by centrifugation for 30 minutes at4000 rpm in a Beckman J-6B. Cell pellets were lysed with 25 ml BugBuster® (Novagen) in the presence of MgCl₂ and DNaseI. Inclusion bodypellets were recovered by centrifugation for 30 minutes at 13000 rpm ina Beckman J2-21 centrifuge. Three detergent washes were then carried outto remove cell debris and membrane components. Each time the inclusionbody pellet was homogenised in a Triton buffer (50 mM Tris-HCl pH 8.0,0.5% Triton-X100, 200 mM NaCl, 10 mM NaEDTA) before being pelleted bycentrifugation for 15 minutes at 13000 rpm in a Beckman J2-21. Detergentand salt was then removed by a similar wash in the following buffer: 50mM Tris-HCl pH 8.0, 1 mM NaEDTA. Finally, the inclusion bodies weredivided into 30 mg aliquots and frozen at −70° C. Inclusion body proteinyield was quantified by solubilising with 6 M guanidine-HCl and an ODmeasurement was taken on a Hitachi U-2001 Spectrophotometer. The proteinconcentration was then calculated using the extinction coefficient.

Approximately 15 mg of TCR β chain and 15 mg of TCR α chain solubilisedinclusion bodies were thawed from frozen stocks and diluted into 10 mlof a guanidine solution (6 M Guanidine-hydrochloride, 50 mM Tris HCl pH8.1, 100 mM NaCl, 10 mM EDTA, 10 mM DTT), to ensure complete chaindenaturation. The guanidine solution containing fully reduced anddenatured TCR chains was then injected into 0.5 litre of the followingrefolding buffer: 100 mM Tris pH 8.1, 400 mM L-Arginine, 2 mM EDTA, 5 MUrea. The redox couple (cysteamine hydrochloride and cystaminedihydrochloride) to final concentrations of 6.6 mM and 3.7 mMrespectively, were added approximately 5 minutes before addition of thedenatured TCR chains. The solution was left for ˜30 minutes. Therefolded TCR was dialysed in Spectra/Por® 1 membrane (Spectrum; ProductNo. 132670) against 10 L H₂O for 18-20 hours. After this time, thedialysis buffer was changed twice to fresh 10 mM Tris pH 8.1 (10 L) anddialysis was continued at 5° C.±3° C. for another ˜8 hours.

Soluble TCR was separated from degradation products and impurities byloading the dialysed refold onto a POROS® 50HQ anion exchange column andeluting bound protein with a gradient of 0-500 mM NaCl in 10 mM Tris pH8.1 over 50 column volumes using an Akta® purifier (GE Healthcare). Peakfractions were pooled and a cocktail of protease inhibitors (Calbiochem)were added. The pooled fractions were then stored at 4° C. and analysedby Coomassie-stained SDS-PAGE before being pooled and concentrated.Finally, the soluble TCR was purified and characterised using a GEHealthcare Superdex® 75HR gel filtration column pre-equilibrated in PBSbuffer (Sigma). The peak eluting at a relative molecular weight ofapproximately 50 kDa was pooled and concentrated prior tocharacterisation by BIAcore® surface plasmon resonance analysis.

Example 3

Binding Characterisation

BIAcore Analysis

A surface plasmon resonance biosensor (BIAcore® 3000) can be used toanalyse the binding of a soluble TCR to its peptide-MHC ligand. This isfacilitated by producing soluble biotinylated peptide-HLA (“pHLA”)complexes which can be immobilised to a streptavidin-coated bindingsurface (sensor chip). The sensor chips may comprise four individualflow cells which enable simultaneous measurement of T-cell receptorbinding to four different pHLA complexes. Manual injection of pHLAcomplex allows the precise level of immobilised class I molecules to bemanipulated easily.

Biotinylated class I HLA-A*02 molecules were refolded in vitro frombacterially-expressed inclusion bodies containing the constituentsubunit proteins and synthetic peptide, followed by purification and invitro enzymatic biotinylation (O'Callaghan et al. (1999) Anal. Biochem.266: 9-15). HLA-A*02-heavy chain was expressed with a C-terminalbiotinylation tag which replaces the transmembrane and cytoplasmicdomains of the protein in an appropriate construct. Inclusion bodyexpression levels of ˜75 mg/litre bacterial culture were obtained. TheMHC light-chain or β2-microglobulin was also expressed as inclusionbodies in E. coli from an appropriate construct, at a level of ˜500mg/litre bacterial culture.

E. coli cells were lysed and inclusion bodies were purified toapproximately 80% purity. Protein from inclusion bodies was denatured in6 M guanidine-HCl, 50 mM Tris pH 8.1, 100 mM NaCl, 10 mM DTT, 10 mMEDTA, and was refolded at a concentration of 30 mg/litre heavy chain, 30mg/litre β2m into 0.4 M L-Arginine, 100 mM Tris pH 8.1, 3.7 mM cystaminedihydrochloride, 6.6 mM cysteamine hydrochloride, 4 mg/L of the AFPpeptide required to be loaded by the HLA-A*02 molecule, by addition of asingle pulse of denatured protein into refold buffer at <5° C. Refoldingwas allowed to reach completion at 4° C. for at least 1 hour.

Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Theprotein solution was then filtered through a 1.5 μm cellulose acetatefilter and loaded onto a POROS® 50HQ anion exchange column (8 ml bedvolume). Protein was eluted with a linear 0-500 mM NaCl gradient in 10mM Tris pH 8.1 using an Akta® purifier (GE Healthcare). HLA-A*02-peptidecomplex eluted at approximately 250 mM NaCl, and peak fractions werecollected, a cocktail of protease inhibitors (Calbiochem) was added andthe fractions were chilled on ice.

Biotinylation tagged pHLA molecules were buffer exchanged into 10 mMTris pH 8.1, 5 mM NaCl using a GE Healthcare fast desalting columnequilibrated in the same buffer. Immediately upon elution, theprotein-containing fractions were chilled on ice and protease inhibitorcocktail (Calbiochem) was added. Biotinylation reagents were then added:1 mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgCl₂, and 5 μg/ml BirAenzyme (purified according to O'Callaghan et al. (1999) Anal. Biochem.266: 9-15). The mixture was then allowed to incubate at room temperatureovernight.

The biotinylated pHLA-A*01 molecules were purified using gel filtrationchromatography. A GE Healthcare Superdex® 75 HR 10/30 column waspre-equilibrated with filtered PBS and 1 ml of the biotinylationreaction mixture was loaded and the column was developed with PBS at 0.5ml/min using an Akta® purifier (GE Healthcare). Biotinylated pHLA-A*02molecules eluted as a single peak at approximately 15 ml. Fractionscontaining protein were pooled, chilled on ice, and protease inhibitorcocktail was added. Protein concentration was determined using aCoomassie-binding assay (PerBio) and aliquots of biotinylated pHLA-A*02molecules were stored frozen at −20° C.

The BIAcore® 3000 surface plasmon resonance (SPR) biosensor measureschanges in refractive index expressed in response units (RU) near asensor surface within a small flow cell, a principle that can be used todetect receptor ligand interactions and to analyse their affinity andkinetic parameters. The BIAcore® experiments were performed at atemperature of 25° C., using PBS buffer (Sigma, pH 7.1-7.5) as therunning buffer and in preparing dilutions of protein samples.Streptavidin was immobilised to the flow cells by standard aminecoupling methods. The pHLA complexes were immobilised via the biotintag. The assay was then performed by passing soluble TCR over thesurfaces of the different flow cells at a constant flow rate, measuringthe SPR response in doing so.

Equilibrium Binding Constant

The above BIAcore® analysis methods were used to determine equilibriumbinding constants. Serial dilutions of the disulfide linked solubleheterodimeric form of the reference AFP TCR were prepared and injectedat constant flow rate of 5 μl min⁻¹ over two different flow cells; onecoated with ˜1000 RU of specific HLA-A*02 complex, the second coatedwith ˜1000 RU of non-specific HLA-A2-peptide complex. Response wasnormalised for each concentration using the measurement from the controlcell. Normalised data response was plotted versus concentration of TCRsample and fitted to a non-linear curve fitting model in order tocalculate the equilibrium binding constant, K_(D) (Price & Dwek,Principles and Problems in Physical Chemistry for Biochemists (2^(nd)Edition) 1979, Clarendon Press, Oxford). The disulfide linked solubleform of the reference AFP TCR (Example 2) demonstrated a K_(D) ofapproximately 754 μM. From the same BIAcore® data the T½ wasapproximately <0.5 s.

Kinetic Parameters

The above BIAcore® analysis methods were also used to determineequilibrium binding constants and off-rates.

For high affinity TCRs (see Example 4 below) K_(D) was determined byexperimentally measuring the dissociation rate constant, k_(off), andthe association rate constant, k_(on). The equilibrium constant K_(D)was calculated as k_(off)/k_(on).

TCR was injected over two different cells one coated with ˜1000 RU ofFMNKFIYEI HLA-A*02 complex, the second coated with ˜1000 RU ofnon-specific HLA-A2-peptide complex. Flow rate was set at 50 μl/min.Typically 250 μl of TCR at ˜1 μM concentration was injected. Buffer wasthen flowed over until the response had returned to baseline or >2 hourshad elapsed. Kinetic parameters were calculated using BIAevaluation®software. The dissociation phase was fitted to a single exponentialdecay equation enabling calculation of half-life.

Example 4

Preparation of Mutated TCRs of the Invention

Phage display is one means by which libraries of AFP TCR variants can begenerated in order to identify higher affinity mutants. The TCR phagedisplay and screening methods described in (Li et al, (2005) NatureBiotech 23 (3): 349-354) were applied to the parental AFP TCR of Example1.

TCRs with improved binding compared to the parental AFP TCR wereidentified, having one or more mutations in the alpha chain variabledomain amino acid residues 31Q, 32S, 94D, 95S, 96G, 97Y, and 98A (usingthe numbering shown in SEQ ID No: 2). Specific examples of the aminoacid sequences of the variable regions of the alpha chains (SEQ ID Nos:6 to 20) of higher affinity TCRs are shown in FIG. 5. These alpha chainsare mutated in CDR1 and/or CDR3.

Expression plasmids containing the TCR α-chain and β-chain respectivelyfor the following TCRs of the invention were prepared as in Example 1:

TCR ID Alpha Chain SEQ ID No Beta Chain SEQ ID No Parental 2 3 ADB327 63 ADB329 7 3 ADB330 8 3 ADB331 9 3 ADB328 10 3 ADB352 11 3 ADB350 12 3ADB332 13 3 ADB351 14 3 ADB349 15 3 ADB353 16 3 ADB326 17 3 ADB333 18 3ADB334 19 3 ADB335 20 3

The plasmids were transformed separately into E. coli strain BL21pLysS,and single ampicillin-resistant colonies grown at 37° C. in TYP(ampicillin 100 μg/ml) medium to OD₆₀₀ of ˜0.6-0.8 before inducingprotein expression with 0.5 mM IPTG. Cells were harvested three hourspost-induction by centrifugation for 30 minutes at 4000 rpm in a BeckmanJ-6B. Cell pellets were lysed with 25 ml Bug Buster® (Novagen) in thepresence of MgCl₂ and DNaseI. Inclusion body pellets were recovered bycentrifugation for 30 minutes at 13000 rpm in a Beckman J2-21centrifuge. Three detergent washes were then carried out to remove celldebris and membrane components. Each time the inclusion body pellet washomogenised in a Triton buffer (50 mM Tris-HCl pH 8.0, 0.5% Triton-X100,200 mM NaCl, 10 mM NaEDTA) before being pelleted by centrifugation for15 minutes at 13000 rpm in a Beckman J2-21. Detergent and salt was thenremoved by a similar wash in the following buffer: 50 mM Tris-HCl pH8.0, 1 mM NaEDTA. Finally, the inclusion bodies were divided into 30 mgaliquots and frozen at −70° C. Inclusion body protein yield wasquantified by solubilising with 6 M guanidine-HCl and an OD measurementwas taken on a Hitachi U-2001 Spectrophotometer. The proteinconcentration was then calculated using the extinction coefficient.

Approximately 10 mg of TCR β chain and 10 mg of TCR α chain solubilisedinclusion bodies for each TCR of the invention were diluted into 10 mlof a guanidine solution (6 M Guanidine-hydrochloride, 50 mM Tris HCl pH8.1, 100 mM NaCl, 10 mM EDTA, 10 mM DTT), to ensure complete chaindenaturation. The guanidine solution containing fully reduced anddenatured TCR chains was then injected into 0.5 litre of the followingrefolding buffer: 100 mM Tris pH 8.1, 400 mM L-Arginine, 2 mM EDTA, 5 MUrea. The redox couple (cysteamine hydrochloride and cystaminedihydrochloride) to final concentrations of 6.6 mM and 3.7 mMrespectively, were added approximately 5 minutes before addition of thedenatured TCR chains. The solution was left for ˜30 minutes. Therefolded TCR was dialysed in Spectra/Por® 1 membrane (Spectrum; ProductNo. 132670) against 10 L H₂O for 18-20 hours. After this time, thedialysis buffer was changed twice to fresh 10 mM Tris pH 8.1 (10 L) anddialysis was continued at 5° C.±3° C. for another ˜8 hours.

Soluble TCR was separated from degradation products and impurities byloading the dialysed refold onto a POROS® 50HQ anion exchange column andeluting bound protein with a gradient of 0-500 mM NaCl in 10 mM Tris pH8.1 over 15 column volumes using an Akta® purifier (GE Healthcare). Thepooled fractions were then stored at 4° C. and analysed byCoomassie-stained SDS-PAGE before being pooled and concentrated.Finally, the soluble TCRs were purified and characterised using a GEHealthcare Superdex® 75HR gel filtration column pre-equilibrated in PBSbuffer (Sigma). The peak eluting at a relative molecular weight ofapproximately 50 kDa was pooled and concentrated prior tocharacterisation by BIAcore® surface plasmon resonance analysis.

The affinity profiles of the thus-prepared TCRs for the AFP epitope wereassessed using the method of Example 3, and compared with the referenceTCR. The results are set forth in the following table:

TCR alpha chain extracellular domain T½ (s) KD μM Parental <0.5 754ADB327 <0.5 489 ADB329 <0.5 356 ADB330 <0.5 178 ADB331 <0.5 79.5 ADB328<0.5 33.3 ADB352 <0.5 20.1 ADB350 0.8 11.0 ADB332 0.7 10.6 ADB351 0.88.0 ADB349 1.8 4.55 ADB353 1.5 4.17 ADB326 1.5 1.50 ADB333 3.8 0.71ADB334 4.3 0.52 ADB335 13.9 0.31

Example 5

Transfection of T-Cells with Parental and Variant AFP TCRs

(a) Lentiviral Vector Preparation by Express-in Mediated TransientTransfection of 293T Cells

A 3rd generation lentiviral packaging system was used to packagelentiviral vectors containing the gene encoding the desired TCR. 293Tcells were transfected with 4 plasmids (one lentiviral vector containingthe TCR alpha chain-P2A-TCR beta chain single ORF gene described inExample 5c (below), and 3 plasmids containing the other componentsnecessary to construct infective but non-replicative lentiviralparticles) using Express-In mediated transfection (Open Biosystems).

For transfection take one T150 flask of 293T cells in exponential growthphase, with cells evenly distributed on the plate, and slightly morethan 50% confluent. Bring Express-In aliquots to room temperature. Place3 ml Serum-Free Medium (RPMI 1640+10 mM HEPES) in a sterile 15 mlconical tube. Add 174 μl of Express-In Reagent directly into theSerum-Free Medium (this provides for a 3.6:1 weight ratio of Reagent toDNA). Mix thoroughly by inverting tubes 3-4 times and incubate at roomtemperature for 5-20 minutes.

In a separate 1.5 ml microtube, add 15 μg plasmid DNA to premixedpackaging mix aliquots (containing 18 μg pRSV.REV (Rev expressionplasmid), 18 μg pMDLg/p.RRE (Gag/Pol expression plasmid), 7 μg pVSV-G(VSV glycoprotein expression plasmid), usually ˜22 and pipette up anddown to ensure homogeneity of the DNA mix. Add ˜1 ml ofExpress-In/Serum-Free Medium to the DNA mix drop wise then pipette upand down gently before transferring back to the remainder of theExpress-In/Serum-Free Medium. Invert tube 3-4 times and incubate at roomtemperature for 15-30 minutes.

Remove old culture medium from flask of cells. Add Express-In/medium/DNA(3 ml) complex to flask direct into the bottom of an upright flask of293T cells. Slowly, place the flask flat to cover the cells and verygently rock the flask to ensure even distribution. After 1 minute add 22ml fresh culture medium (R10+HEPES: RPMI 1640, 10% heat-inactivated FBS,1% Pen/Strep/L-glutamine, 10 mM HEPES) and carefully return toincubator. Incubate overnight at 37° C./5% CO2. After 24 hours, proceedto harvest the medium containing packaged lentiviral vectors.

To harvest the packaged lentiviral vectors, filter the cell culturesupernatent through a 0.45 micron nylon syringe filter, centrifuge theculture medium at 10,000 g for 18 hours (or 112,000 g for 2 hours),remove most of the supernatant (taking care not to disturb the pellet)and resuspend the pellet in the remaining few ml of supernatant (usuallyabout 2 ml from a 31 ml starting volume per tube). Snap freeze on dryice in 1 ml aliquots and store at −80° C.

(b) Transduction of T Cells with Packaged Lentiviral Vectors ContainingGene of Interest

Prior to transduction with the packaged lentiviral vectors, human Tcells (CD8 or CD4 or both depending on requirements) are isolated fromthe blood of healthy volunteers. These cells are counted and incubatedovernight in R10 containing 50 U/ml IL-2 at 1×10⁶ cells per ml (0.5ml/well) in 48 well plates with pre-washed anti-CD3/CD28 antibody-coatedmicrobeads (Dynabeads® T cell expander, Invitrogen) at a ratio of 3beads per cell.

After overnight stimulation, 0.5 ml of neat packaged lentiviral vectoris added to the desired cells. Incubate at 37° C./5% CO2 for 3 days. 3days post-transduction count cells and dilute to 0.5×10⁶ cells/ml. Addfresh medium containing IL-2 as required. Remove beads 5-7 dayspost-transduction. Count cells and replace or add fresh mediumcontaining IL-2 at 2 day intervals. Keep cells between 0.5×10⁶ and 1×10⁶cells/ml. Cells can be analysed by flow cytometry from day 3 and usedfor functional assays (e.g. ELISpot for IFNγ release, see Example 6)from day 5. From day 10, or when cells are slowing division and reducedin size, freeze cells in aliquots of at least 4×10⁶ cells/vial (at 1×10⁷cells/ml in 90% FBS/10% DMSO) for storage.

(c) Parental TCR Gene for T-Cell Transfection by Methods (a) and (b)Above

FIG. 7 is a DNA sequence (SEQ ID No: 23) encoding the parental AFP TCR(codon-optimised for maximal human cell expression). It is a full lengthalpha chain—Porcine teschovirus-1 2A—full length beta chain single openreading frame construct. The 2A sequence is underlined, and is precededby nucleotides encoding a furin cleavage site to assist proteolyticremoval of the 2A sequence (discussed further below in relation to FIG.8 (SEQ ID No: 24). Peptide bond skipping during protein translation ofthe mRNA at the 3′ end of the 2A sequence produces two proteins: 1)alpha TCR chain-2A fusion, 2) beta TCR chain. SEQ ID No: 23 includesNheI and SalI restriction sites (underlined).

FIG. 8 is the amino acid sequence (SEQ ID No: 24) corresponding to FIG.7

In FIG. 8:

-   -   M1-Q22 is a leader sequence which is removed on maturation of        the parental alpha chain TCR;    -   Q23-S274 corresponds to the parental alpha chain sequence;    -   Q23-N246 corresponds to the parental alpha chain extracellular        domain;    -   L247-T268 is the alpha chain transmembrane region of the mature        TCR;    -   L269-S274 is the alpha chain intracellular region of the mature        TCR;    -   R277-R280 is the furin cleavage site to assist proteolytic        removal, in the Golgi apparatus, of the P2A sequence A285-P303;    -   G275, S276, S281 to G284, are flexible linkers allowing full        function of the furin cleavage and P2A sequences;    -   R304-V323 is a leader sequence which is removed on maturation of        the parental beta chain TCR;    -   D324-G614 corresponds to the parental beta chain sequence;    -   D324-E585 corresponds to the parental beta chain extracellular        domain;    -   I586-V607 is the beta chain transmembrane region of the mature        TCR;    -   K608-G614 is the beta chain intracellular region of the mature        TCR.

(d) T-Cells Transfected with Parental and High Affinity AFP TCRs

Following the procedures described in (a) and (b) above, the parentalAFP alpha-2A-beta TCR gene (SEQ ID No: 23 (FIG. 7)) was inserted intothe pELNSxv lenti vector using the NheI and SalI restriction sitesunique to both DNA constructs, and transfected T-cells created.

Similarly, T-cells may be created by transfection with genes identicalto SEQ ID No: 23 (FIG. 7) except that they encode an alpha chainvariable domain having one of SEQ ID Nos: 6 to 20 associated with thevariable domain sequence (D1 to T112) of the parental beta chain SEQ IDNo: 3;

Example 6

Activation of AFP TCR Engineered T Cells

The following assay was carried out to demonstrate the activation ofTCR-transduced cytotoxic T lymphocytes (CTLs) in response to tumour celllines. IFN-γ production, as measured using the ELISPOT assay, was usedas a read-out for cytotoxic T lymphocyte (CTL) activation.

ELISPOTs

Reagents

Assay media: 10% FCS (Gibco, Cat#2011-09), 88% RPMI 1640 (Gibco,Cat#42401), 1% glutamine (Gibco Cat#25030) and 1%penicillin/streptomycin (Gibco Cat#15070-063).

Wash buffer: 0.01M PBS/0.05% Tween 20

PBS (Gibco Cat#10010)

The Human IFNγ ELISPOT kit (BD Bioscience; Cat#551849) containingcapture and detection antibodies and Human IFN-γ PVDF ELISPOT 96 wellplates, with associated AEC substrate set (BD Bioscience, Cat#551951)

Methods

Target Cell Preparation

The target cells used in this method were natural epitope-presentingcells: HepG2 hepatocellular carcinoma cells which are both HLA-A2⁺ AFP⁺.HEP2 normal human hepatocytes, which are HLA-A2⁺ AFP⁻, were used as anegative control. Sufficient target cells (50,000 cells/well) werewashed by centrifugation three times at 1200 rpm, 10 min in a Megafuge®1.0 (Heraeus). Cells were then re-suspended in assay media at 10⁶cells/ml.

Effector Cell Preparation

The effector cells (T cells) used in this method were peripheral bloodlymphocytes (PBL), obtained by negative selection using CD14 and CD25microbead kits (Miltenyi Biotech Cat#130-050-201 and 130-092-983respectively) from freshly isolated peripheral blood mononuclear cells(PBMC) from the venous blood of healthy volunteers. Cells werestimulated with antiCD3/CD28 coated beads (Dynabeads® T cell expander,Invitrogen), transduced with lentivirus carrying the gene encoding thefull αβ TCR of interest (based on the construct described in Example 5and shown in FIG. 7) and expanded in assay media containing 50U/ml IL-2until between 10 and 13 days post transduction. These cells were thenplaced in assay media prior to washing by centrifugation at 1200 rpm, 10min in a Megafuge® 1.0 (Heraeus). Cells were then re-suspended in assaymedia at a 4× the final required concentration.

Plates were prepared as follows: 100 μl anti-IFN-γ capture antibody wasdiluted in 10 ml sterile PBS per plate. 100 μl of the diluted captureantibody was then dispensed into each well. The plates were thenincubated overnight at 4° C. Following incubation the plates were washed(programme 1, plate type 2, Ultrawash Plus 96-well plate washer; Dynex)to remove the capture antibody. Plates were then blocked by adding 200μl of assay media to each well and incubated at room temperature for twohours. The assay media was then washed from the plates (programme 1,plate type 2, Ultrawash Plus 96-well plate washer, Dynex) and anyremaining media was removed by flicking and tapping the ELISPOT plateson a paper towel.

The constituents of the assay were then added to the ELISPOT plate inthe following order:

50 μl of target cells 10⁶ cells/ml (giving a total of 50,000 targetcells/well)

50 μl media (assay media)

50 μl effector cells (20,000 TCR-transduced PBL cells/well)

The plates were then incubated overnight (37° C./5% CO2). The next daythe plates were washed three times (programme 1, plate type 2, UltrawashPlus 96-well plate washer, Dynex) with wash buffer and tapped dry onpaper towel to remove excess wash buffer. 100 μl of primary detectionantibody was then added to each well. The primary detection antibody wasdiluted into 10 ml of dilution buffer (the volume required for a singleplate) using the dilution specified in the manufacturer's instructions.Plates were then incubated at room temperature for at least 2 hoursprior to being washed three times (programme 1, plate type 2, UltrawashPlus 96-well plate washer, Dynex) with wash buffer; excess wash bufferwas removed by tapping the plate on a paper towel.

Secondary detection was performed by adding 100 μl of dilutedstreptavidin-HRP to each well and incubating the plate at roomtemperature for 1 hour. The streptavidin-HRP was diluted into 10 mldilution buffer (the volume required for a single plate), using thedilution specified in the manufacturer's instructions. The plates werethen washed three times (programme 1, plate type 2, Ultrawash Plus96-well plate washer, Dynex) with wash buffer and tapped on paper towelto remove excess wash buffer. Plates were then washed twice with PBS byadding 200 μl to each well, flicking the buffer off and tapping on apaper towel to remove excess buffer. No more than 15 mins prior to use,one drop (20 μl) of AEC chromogen was added to each 1 ml of AECsubstrate and mixed. 10 ml of this solution was prepared for each plate;100 μl was added per well. The plate was then protected from light usingfoil, and spot development monitored regularly, usually occurring within5-20 mins. The plates were washed in tap water to terminate thedevelopment reaction, and shaken dry prior to their disassembly intothree constituent parts. The plates were then allowed to dry at roomtemperature for at least 2 hours prior to counting the spots using anImmunospot® Plate reader (CTL; Cellular Technology Limited).

Results

IFNγ release by activated TCR-transduced T cells in response to avariety of AFP-positive and control tumour cell lines was tested byELISPOT assay (as described above). The number of ELISPOT spots observedin each well was plotted using Graph Pad Prism®.

CD4+, CD8+ or mixed CD4+/CD8+ T cells expressing the WT TCR or one ofTCR Nos:1-5 (as described in the table below) were incubated with AFP+HLA:A2+ tumour cell line HepG2 or with AFP-HLA:A2+ HEP2 normalhepatocytes. A sample containing no T cells was used as a control.

TCR α variable domain TCR β variable domain TCR No SEQ ID NO: SEQ ID NO:1 11 D1 to T112 of SEQ ID No: 3 2 12 D1 to T112 of SEQ ID No: 3 3 13 D1to T112 of SEQ ID No: 3 4 14 D1 to T112 of SEQ ID No: 3 5 15 D1 to T112of SEQ ID No: 3

FIG. 9 demonstrates that T cells transduced with the TCRs described inthe table above are activated in response to AFP positive tumour cells(HepG2). Activation of these variant TCRs is greater than for the WTTCR. Activation by AFP negative normal hepatocytes (HEP2) is minimaldemonstrating the specificity of the TCRs for AFP.

The invention is further described by the following numbered paragraphs:

1. A non-naturally occurring and/or purified and/or engineered T cellreceptor (TCR) having the property of binding to FMNKFIYEI (SEQ IDNo: 1) HLA-A2 complex and comprising at least one TCR alpha chainvariable domain and/or at least one TCR beta chain variable domain,

-   -   the alpha chain variable domain comprising an amino acid        sequence that has at least 90% identity to the sequence of amino        acid residues 1-112 of SEQ ID No: 2, and/or    -   the beta chain variable domain comprising an amino acid sequence        that has at least 90% identity to the sequence of amino acid        residues 1-112 of SEQ ID No: 3.

2. The TCR of paragraph 1, wherein the alpha chain variable domainincludes a mutation in one or more of the amino acids corresponding to31Q, 32S, 94D, 95S, 96G, 97Y and 98A.

3. The TCR of paragraph 1 or paragraph 2, wherein the alpha chainvariable domain includes at least one of the following mutations:

Residue no. 31Q F Y 32S A 94D Q 95S N 96G S 97Y V 98A S

4. The TCR of any preceding paragraph, wherein the alpha chain variabledomain comprises an amino acid sequence having at least 90% identity toresidues 1-112 of any one of SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8,SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, SEQ ID No:13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ IDNo: 18, SEQ ID No: 19 and SEQ ID No: 20.

5. The TCR of any preceding paragraph, wherein the alpha chain variabledomain comprises Q1 to H112 of SEQ ID No: 11, SEQ ID No 12 or SEQ ID No13 and/or the beta chain variable domain comprise D1 to T112 of SEQ IDNo: 3.

6. The TCR of paragraph 1, wherein the alpha chain variable domaincomprises amino acid resides 1-112 of SEQ ID No: 2, and the beta chainvariable domain comprises amino acid residues 1-112 of SEQ ID No: 3.

7. The TCR of any preceding paragraph having an alpha chain TRACconstant domain sequence and/or a beta chain TRBC1 or TRBC2 constantdomain sequence.

8. The TCR of paragraph 7, wherein, the alpha and/or beta chain constantdomain sequence(s) are modified by truncation or substitution to deletethe native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 ofexon 2 of TRBC1 or TRBC2.

9. The TCR of paragraph 7 or paragraph 8, wherein the alpha and/or betachain constant domain sequence(s) are modified by substitution ofcysteine residues for Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2, thecysteines forming a disulfide bond between the alpha and beta constantdomains of the TCR.

10. The TCR of any preceding paragraph, which is in single chain formatof the type Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ or Vα-Cα-L-Vβ-Cβwherein Vα and Vβ are TCR α and β variable regions respectively, Cα andCβ are TCR α and β constant regions respectively, and L is a linkersequence.

11. The TCR of any one of paragraphs 1-9, which is an alpha-betaheterodimer.

12. The TCR of any preceding paragraph associated with a detectablelabel, a therapeutic agent or a PK modifying moiety.

13. Non-naturally occurring and/or purified and/or engineered nucleicacid encoding the TCR of any one of the preceding paragraphs.

14. A non-naturally occurring and/or purified and/or engineered cell,especially a T-cell, presenting a TCR of any one of paragraphs 1-12.

15. A cell harbouring

-   -   (a) a TCR expression vector which comprises nucleic acid of        paragraph 13 encoding in a single open reading frame, or two        distinct open reading frames, the alpha chain and the beta chain        respectively; or    -   (b) a first expression vector which comprises nucleic acid        encoding the alpha chain of a TCR of any of paragraphs 1 to 12,        and a second expression vector which comprises nucleic acid        encoding the beta chain of a TCR of any of paragraphs 1 to 12.

16. A pharmaceutical composition comprising a TCR of any one ofparagraphs 1 to 12 or a cell of paragraph 14 or paragraph 15, togetherwith one or more pharmaceutically acceptable carriers or excipients.

17. A non-naturally occurring and/or purified and/or engineered TCRwhich binds the FMNKFIYEI (SEQ ID No: 1) peptide presented as apeptide-HLA-A2 complex, or a cell expressing and/or presenting such aTCR, for use in medicine.

18. The TCR or cell for use of paragraph 17, for use in a method oftreating cancer.

19. The TCR or cell for use of paragraph 18, wherein the methodcomprises adoptive therapy.

20. The TCR or cell for use of any one of paragraphs 17 to 19, whereinthe TCR is in any one of paragraphs 1 to 12 and/or wherein the cell isof paragraph 14 or paragraph 15.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. “A method for treating a patient expressing aFMNKFIYEI (SEQ ID NO: 1)-HLA-A2 complex comprising: (a) administering tothe patient an immune cell comprising a non-naturally occurring and/orpurified and/or engineered T cell receptor (TCR) having the property ofbinding to said FMNKFIYEI (SEQ ID NO: 1) HLA-A2 complex or (b)administering to the patient a non-naturally occurring and/or purifiedand/or engineered T cell receptor (TCR) having the property of bindingto said FMNKFIYEI (SEQ ID NO: 1) HLA-A2 complex wherein said TCR furthercomprises a therapeutic agent, wherein the TCR of (a) or (b) comprisesat least one TCR alpha chain variable domain and at least one TCR betachain variable domain, wherein the alpha chain variable domain comprisesat least one of the following sets of CDR sequences:” CDR1 DRGSQAresidues 27 to 32 of SEQ ID NO: 6 CDR2 IYSNGDresidues 50 to 55 of SEQ ID NO: 6 CDR3 AVNSDSresidues 90 to 101 of SEQ ID NO: 6 GYALNF or CDR1 DRGSQSresidues 27 to 32 of SEQ ID NO: 7 CDR2 IYSNGDresidues 50 to 55 of SEQ ID NO: 7 CDR3 AVNSDSresidues 90 to 101 of SEQ ID NO: 7 SYALNF or CDR1 DRGSQSresidues 27 to 32 of SEQ ID NO: 8 CDR2 IYSNGDresidues 50 to 55 of SEQ ID NO: 8 CDR3 AVNSDSresidues 90 to 101 of SEQ ID NO: 8 GVALNF or CDR1 DRGSQAresidues 27 to 32 of SEQ ID NO: 9 CDR2 IYSNGDresidues 50 to 55 of SEQ ID NO: 9 CDR3 AVNSDSresidues 90 to 101 of SEQ ID NO: 9 GVALNF or CDR1 DRGSQSresidues 27 to 32 of SEQ ID NO: 10 CDR2 IYSNGDresidues 50 to 55 of SEQ ID NO: 10 CDR3 AVNSQSresidues 90 to 101 of SEQ ID NO: 10 GYALNF or CDR1 DRGSQSresidues 27 to 32 of SEQ ID NO: 11 CDR2 IYSNGDresidues 50 to 55 of SEQ ID NO: 11 CDR3 AVNSQSresidues 90 to 101 of SEQ ID NO: 11 GYSLNF or CDR1 DRGSQSresidues 27 to 32 of SEQ ID NO: 12 CDR2 IYSNGDresidues 50 to 55 of SEQ ID NO: 12 CDR3 AVNSQSresidues 90 to 101 of SEQ ID NO: 12 SYALNF or CDR1 DRGSQAresidues 27 to 32 of SEQ ID NO: 13 CDR2 IYSNGDresidues 50 to 55 of SEQ ID NO: 13 CDR3 AVNSQSresidues 90 to 101 of SEQ ID NO: 13 GYALNF or CDR1 DRGSQSresidues 27 to 32 of SEQ ID NO: 14 CDR2 IYSNGDresidues 50 to 55 of SEQ ID NO: 14 CDR3 AVNSQSresidues 90 to 101 of SEQ ID NO: 14 GVALNF or CDR1 DRGSQSresidues 27 to 32 of SEQ ID NO: 15 CDR2 IYSNGDresidues 50 to 55 of SEQ ID NO: 15 CDR3 AVNSQNresidues 90 to 101 of SEQ ID NO: 15 GYALNF or CDR1 DRGSFSresidues 27 to 32 of SEQ ID NO: 16 CDR2 IYSNGDresidues 50 to 55 of SEQ ID NO: 16 CDR3 AVNSDSresidues 90 to 101 of SEQ ID NO: 16 GYALNF or CDR1 DRGSYSresidues 27 to 32 of SEQ ID NO: 17 CDR2 IYSNGDresidues 50 to 55 of SEQ ID NO: 17 CDR3 AVNSDSresidues 90 to 101 of SEQ ID NO: 17 GYALNF or CDR1 DRGSYSresidues 27 to 32 of SEQ ID NO: 18 CDR2 IYSNGDresidues 50 to 55 of SEQ ID NO: 18 CDR3 AVNSDSresidues 90 to 101 of SEQ ID NO: 18 SYALNF or CDR1 DRGSYSresidues 27 to 32 of SEQ ID NO: 19 CDR2 IYSNGDresidues 50 to 55 of SEQ ID NO: 19 CDR3 AVNSDSresidues 90 to 101 of SEQ ID NO: 19 GVALNF or CDR1 DRGSYSresidues 27 to 32 of SEQ ID NO: 20 CDR2 IYSNGDresidues 50 to 55 of SEQ ID NO: 20 CDR3 AVNSQSresidues 90 to 101 of SEQ ID NO: 20 GYALNF

and the wherein the beta chain variable domain comprises the followingset of CDR sequences: CDR1 SGDLS residues 27 to 31 of SEQ ID NO: 5 CDR2YYNGEE residues 49 to 54 of SEQ ID NO: 5 CDR3 ASSLGGresidues 92 to 102 of SEQ ID NO: 5 ESEQF

wherein the TCR further comprises a therapeutic agent or is expressed inan immune cell.
 2. The method of claim 1 wherein the treating istreating cancer.
 3. The method of claim 2 wherein the treating cancer istreating hepatocellular carcinoma.
 4. The method of claim 1 wherein thetreating comprises treating hepatocellular carcinoma.
 5. The method ofclaim 1 wherein the treating comprises adoptive therapy.
 6. The methodof claim 1, wherein the treating comprises treating alpha Fetoprotein(AFP) positive tumor cells.